We have employed vaccinia virus as a vector to express various portions of the dengue order to study proteolytic processing in the nonstructural protein (NS) region. This study was carried out in part to test the processing model proposed recently predicting that NS3 is a protease based on limited sequence homology to serine proteases. Indirect evidence showing that NS3 can act in trans to cleave NS2A/NS2B and NS2B/NS3 was obtained from co-infection experiments using vNS2A-NS2B and vNS2A-deltaNS2B(10)-30%NS3 or vNS2A-deltaNS2B(20)-30%NS3 in which full length NS2B was produced. However, co-infections using vNS2A-NS2B or vNS2A-NS2B-12%NS3 and vNS3 or v3O%NS3 did not demonstrate efficient trans-acting activity of NS3. The reason for this discrepancy is not known. Our results demonstrate that NS2B is also required for these NS cleavages. NS2B acts in trans to facilitate NS2B/NS3 and NS2A/NS2B cleavage. Thus, both NS2B and NS3 together appear to be required for a protease activity that cleaves NS2A/NS2B and NS2B/NS3. In addition, the protease activity presumably is also responsible for the NS3/NS4A and NS4B/NS5 cleavages. Our results also showed that NS2B is needed for the NS3/NS4A cleavage. This activity of NS2B is either cis-acting or very inefficiently trans-acting.